The expression of survivin positively correlates with cancer drug resistance and poor patient survival. Survivin is ubiquitously expressed in most types of cancer, but has very low expression in normal tissues. Thus, developing novel selective survivin inhibitors as potential cancer therapies is highly significant. We recently discovered a new scaffold provided by compound UC-112. UC-112 strongly inhibits cancer cell proliferation, selectively degrades survivin among other IAPs, and potently suppresses melanoma tumor growth in vivo. Based on these preliminary studies, our overall hypothesis is that the novel scaffold of UC-112 is exquisitely selective for survivin. The objective of this project is to optimize the UC-112 scaffold using integrated structural biology, molecular modeling, medicinal chemistry, and molecular biology approaches. Aim 1. Perform computer-aided drug design based on the UC-112 scaffold and iteratively optimize the anticancer activity. Aim 1.1: Using the crystal structure of survivin-Smac complex and our predictive molecular models for the UC-112 scaffold, we will design and synthesize focused sets of UC-112 analogs to optimize their activity and elucidate the structure-activity relationships. Aim 1.2: Screen the new compounds using a panel of human melanoma cell lines and three-dimensional colony formation assays to identify up to 20 best UC-112 analogs (criteria: IC50 < 100 nM and can overcome multidrug resistance) to be advanced to Aim 2. Aim 2. Define the biological mechanism(s) of action of newly developed UC-112 analogs. Aim 2.1: Structural characterization of UC-112 analogs interacting with survivin and other IAPs. We will confirm the on-target survivin inhibition and selectivity among other IAPs using ITC and SPR measurements. We will also solve the X-ray crystal structures of survivin in complex with potent UC-112 analogs. This information will feed back to Aim 1 to optimize the molecular models for more efficient structural optimization. Aim 2.2: Evaluation of the effect of UC-112 on survivin stability and/or blockage between survivin and caspases using ubiquitination, pulse-chase, and Western blot analyses. Aim 2.3: Investigation of whether UC-112 analogs induce differentiated degrees of apoptosis in cancer cells with distinct survivin expression levels. Aim 2.4: Determination of any off-target effects of the new analogs and selectivity among other IAPs or additionally potential targets of the new UC-112 analogs using pulldowns and antibody array analysis. Aim 3. Determine the anticancer activity of selective survivin inhibitors in vivo. Aim 3.1: Evaluate compound stability, pharmacokinetics (PK), and pharmacodynamics (PD) properties to select up to six best UC-112 analogs for subsequent in vivo efficacy studies. Aim 3.2: Evaluate anticancer activities of the selected survivin inhibitors against human melanoma tumor growth in vivo (in mice) and the potential toxicity to normal cells/tissues. Aim 3.3: Evaluate the ability of our selective survivin inhibitors to treat melanoma metastasis in vivo usin our established experimental lung metastasis model.